Preserving enhancement in freeze-dried contrast agent ST68: Examination of excipients

The perfluorcarbon (perfluorobutane) ultrasound contrast agent ST68, composed of sonicated mixtures of non-ionic surfactants, is stable in solution for only a few weeks at 4 °C. Freeze-drying critically diminished ST68's ability to reflect ultrasound (its echogenicity). A method of incorporating specific lyoprotectants before lyophilization was investigated. Reintroduction of perfluorobutane to the protected freeze-dried sample, followed by reconstituting with water preserved echogenicity. Glucose, trehalose, sucrose, and mannitol were tested at 100 mM and in vitro echogenicity data was collected from samples with dose concentrations of 50–300 μl/l. Glucose was found to be the best lyoprotectant providing an average (n = 3) maximum peak enhancement of 23.2 ± 1.2 dB in vitro, measured at 5 MHz, 684 kPa, and a pulse repetition frequency (PRF) of 100 Hz (p < 0.05 over freeze-dried ST68 control) and 20.8 ± 0.8 dB in vivo in New Zealand white rabbits at 5 MHz and a PRF of 6.7 kHz. Pulse inversion harmonic US images of a rabbit kidney, pre- and post-contrast injection (0.1 ml/kg), showed excellent enhancement and clear vascular delineation, similar to that of the original agent. For the first time this contrast agent can be successfully freeze-dried yielding a longer self-life without the need for refrigeration.